Model Of Transient Focal Ischemia Research Articles

SLC22A17 as a Cell Death-Linked Regulator of Tight Junctions in Cerebral Ischemia.

Beyond neuronal injury, cell death pathways may also contribute to vascular injury after stroke. We examined protein networks linked to major cell death pathways and identified SLC22A17 (solute carrier family 22 member 17) as a novel mediator that regulates endothelial tight junctions after ischemia and inflammatory stress. Protein-protein interactions and brain enrichment analyses were performed using STRING, Cytoscape, and a human tissue-specific expression RNA-seq database. In vivo experiments were performed using mouse models of transient focal cerebral ischemia. Human stroke brain tissues were used to detect SLC22A17 by immunostaining. In vitro experiments were performed using human brain endothelial cultures subjected to inflammatory stress. Immunostaining and Western blot were used to assess responses in SLC22A17 and endothelial tight junctional proteins. Water content, dextran permeability, and electrical resistance assays were used to assess edema and blood-brain barrier (BBB) integrity. Gain and loss-of-function studies were performed using lentiviral overexpression of SLC22A17 or short interfering RNA against SLC22A17, respectively. Protein-protein interaction analysis showed that core proteins from apoptosis, necroptosis, ferroptosis, and autophagy cell death pathways were closely linked. Among the 20 proteins identified in the network, the iron-handling solute carrier SLC22A17 emerged as the mediator enriched in the brain. After cerebral ischemia in vivo, endothelial expression of SLC22A17 increases in both human and mouse brains along with BBB leakage. In human brain endothelial cultures, short interfering RNA against SLC22A17 prevents TNF-α (tumor necrosis factor alpha)-induced ferroptosis and downregulation in tight junction proteins and disruption in transcellular permeability. Notably, SLC22A17 could repress the transcription of tight junctional genes. Finally, short interfering RNA against SLC22A17 ameliorates BBB leakage in a mouse model of focal cerebral ischemia. Using a combination of cell culture, human stroke samples, and mouse models, our data suggest that SLC22A17 may play a role in the control of BBB function after cerebral ischemia. These findings may offer a novel mechanism and target for ameliorating BBB injury and edema after stroke.

Impact of inflammatory preconditioning on murine microglial proteome response induced by focal ischemic brain injury.

Preconditioning with lipopolysaccharide (LPS) induces neuroprotection against subsequent cerebral ischemic injury, mainly involving innate immune pathways. Microglia are resident immune cells of the central nervous system (CNS) that respond early to danger signals through memory-like differential reprogramming. However, the cell-specific molecular mechanisms underlying preconditioning are not fully understood. To elucidate the distinct molecular mechanisms of preconditioning on microglia, we compared these cell-specific proteomic profiles in response to LPS preconditioning and without preconditioning and subsequent transient focal brain ischemia and reperfusion, - using an established mouse model of transient focal brain ischemia and reperfusion. A proteomic workflow, based on isolated microglia obtained from mouse brains by cell sorting and coupled to mass spectrometry for identification and quantification, was applied. Our data confirm that LPS preconditioning induces marked neuroprotection, as indicated by a significant reduction in brain infarct volume. The established brain cell separation method was suitable for obtaining an enriched microglial cell fraction for valid proteomic analysis. The results show a significant impact of LPS preconditioning on microglial proteome patterns by type I interferons, presumably driven by the interferon cluster regulator proteins signal transducer and activator of transcription1/2 (STAT1/2).

Plasma metabonomic study on the effect of Para‑hydroxybenzaldehyde intervention in a rat model of transient focal cerebral ischemia.

Gastrodiaelata Blume has been widely used to treat various central and peripheral nerve diseases, and Para‑hydroxybenzaldehyde (PHBA) is one of the indicated components suggested to provide a neuroprotective effect. In our previous, it was shown that PHBA protected mitochondria against cerebral ischemia‑reperfusion (I/R) injury in rats. In the present study, how PHBA regulated the metabolic mechanism in blood following cerebral I/R was assessed to identify an effective therapeutic target for the prevention and treatment of ischemic stroke (IS). First, a rat model of cerebral ischemia‑reperfusion injury was established via middle cerebral artery occlusion/reperfusion (MCAO/R). The therapeutic effect of PHBA on brain I/R was evaluated by assessing the neurological function score, triphenyl tetrazolium chloride, hematoxylin and eosin, and Nissl staining. Next, a non‑targeted metabolomic based on high‑performance liquid chromatography quadrupole time‑of‑flight mass spectrometry was established to identify differential metabolites. Finally, a targeted metabolic spectrum was analyzed and the potential therapeutic targets were verified by Western blotting. The results showed that the neurological function score, cerebral infarction area, hippocampal morphology, and the number of neurons in the PHBA group were significantly improved compared with the model group. Metabonomic analysis showed that 13 different metabolites were identified between the model and PHBA group, which may be involved in the 'tricarboxylic acid cycle', 'glutathione metabolism', and 'mutual transformation of pentose and glucuronates', amongst others. Among these, the levels of the most significant differential metabolite, dGMP, decreased significantly following PHBA treatment. Western blotting was used to verify the expression of membrane‑associated guanosine kinase PSD‑95 and the subunit of glutamate AMPA receptor GluA1, which significantly increased after PHBA treatment. In addition, it was also found that PHBA increased the expression of the light chain‑3 protein and autophagy effector protein 1, whilst the expression of sequestosome‑1 decreased, indicating that PHBA promoted autophagy. Similarly, in TUNEL staining and detection of apoptosis‑related proteins, it was found that MCAO/R upregulated the expression of Bax and cleaved‑caspase‑3 whilst downregulating the expression of Bcl‑2 and increasing the apoptosis of hippocampal neurons; PHBA reversed this situation. These results suggest that cerebral I/R causes postsynaptic dysfunction by disrupting the interaction between PSD‑95 and AMPARs, and the inhibition of the autophagy system eventually leads to the apoptosis of hippocampal neurons.

Neuroprotective Effects of Tryptanthrin-6-Oxime in a Rat Model of Transient Focal Cerebral Ischemia.

The activation of c-Jun N-terminal kinase (JNK) plays an important role in stroke outcomes. Tryptanthrin-6-oxime (TRYP-Ox) is reported to have high affinity for JNK and anti-inflammatory activity and may be of interest as a promising neuroprotective agent. The aim of this study was to investigate the neuroprotective effects of TRYP-Ox in a rat model of transient focal cerebral ischemia (FCI), which involved intraluminal occlusion of the left middle cerebral artery (MCA) for 1 h. Animals in the experimental group were administered intraperitoneal injections of TRYP-Ox 30 min before reperfusion and 23 and 47 h after FCI. Neurological status was assessed 4, 24, and 48 h following FCI onset. Treatment with 5 and 10 mg/kg of TRYP-Ox decreased mean scores of neurological deficits by 35-49 and 46-67% at 24 and 48 h, respectively. At these doses, TRYP-Ox decreased the infarction size by 28-31% at 48 h after FCI. TRYP-Ox (10 mg/kg) reduced the content of interleukin (IL) 1β and tumor necrosis factor (TNF) in the ischemic core area of the MCA region by 33% and 38%, respectively, and attenuated cerebral edema by 11% in the left hemisphere, which was affected by infarction, and by 6% in the right, contralateral hemisphere 24 h after FCI. TRYP-Ox reduced c-Jun phosphorylation in the MCA pool at 1 h after reperfusion. TRYP-Ox was predicted to have high blood-brain barrier permeability using various calculated descriptors and binary classification trees. Indeed, reactive oxidant production was significantly lower in the brain homogenates from rats treated with TRYP-Ox versus that in control animals. Our data suggest that the neuroprotective activity of TRYP-Ox may be due to the ability of this compound to inhibit JNK and exhibit anti-inflammatory and antioxidant activity. Thus, TRYP-Ox may be considered a promising neuroprotective agent that potentially could be used for the development of new treatment strategies in cerebral ischemia.

Temporal patterns and distribution of pyroptosis-related molecules and effects of human mesenchymal stem cells on pyroptosis following cerebral ischemia/reperfusion in rats

ObjectivesPyroptosis is a new type of programmed cell death that has a strong proinflammatory effect. The present study investigated the dynamic changes of pyroptosis-related molecules and the effect of mesenchymal stem cells (MSCs) on pyroptosis following cerebral ischemia/reperfusion (I/R). Materials and MethodsThe temporal pattern and cellular distribution of caspase-1, Gasdermin D and E (GSDMD and GSDME) in the peri-infarct area, and the effect of human MSCs on GSDMD, IL-1β, IL-18, Lactate dehydrogenase (LDH) and neurological function were studied in a rat model of transient focal cerebral ischemia. ResultsThe expression of caspase-1 mRNA increased with time, with a protein level of pro-caspase-1 comparable to its mRNA level, while the level of cleaved-caspase-1 protein peaked at 48 h following I/R. Increased levels of GSDMD mRNA and protein were also observed, with a peak level at 24 h. There were no significant changes in GSDME mRNA or protein expression after I/R. In regards to changes in the number of cells expressing GSDMD after I/R, that for neurons was more significant than those for microglia and astrocytes. The modified neurological severity score discrepancy and the expression of GSDMD showed no significant differences within 24 h following I/R between the MSC- and NS-treated groups, but MSCs treatment promoted the secretion of IL-1β, IL-18 and LDH. ConclusionsIn the early stage of cerebral infarction in rats, there were dynamic changes in pyroptosis-related molecules (caspase-1 and GSDMD), but MSCs showed no effect on the levels of GSDMD or neurological function.

Embracing Heterogeneity in The Multicenter Stroke Preclinical Assessment Network (SPAN) Trial.

The Stroke Preclinical Assessment Network (SPAN) is a multicenter preclinical trial platform using rodent models of transient focal cerebral ischemia to address translational failure in experimental stroke. In addition to centralized randomization and blinding and large samples, SPAN aimed to introduce heterogeneity to simulate the heterogeneity embodied in clinical trials for robust conclusions. Here, we report the heterogeneity introduced by allowing the 6 SPAN laboratories to vary most of the biological and experimental model variables and the impact of this heterogeneity on middle cerebral artery occlusion (MCAo) performance. We included the modified intention-to-treat population of the control mouse cohort of the first SPAN trial (n=421) and examined the biological and procedural independent variables and their covariance. We then determined their impact on the dependent variables cerebral blood flow drop during MCAo, time to achieve MCAo, and total anesthesia duration using multivariable analyses. We found heterogeneity in biological and procedural independent variables introduced mainly by the site. Consequently, all dependent variables also showed heterogeneity among the sites. Multivariable analyses with the site as a random effect variable revealed filament choice as an independent predictor of cerebral blood flow drop after MCAo. Comorbidity, sex, use of laser Doppler flow to monitor cerebral blood flow, days after trial onset, and maintaining anesthesia throughout the MCAo emerged as independent predictors of time to MCAo. Total anesthesia duration was predicted by most independent variables. We present with high granularity the heterogeneity introduced by the biological and model selections by the testing sites in the first trial of cerebroprotection in rodent transient filament MCAo by SPAN. Rather than trying to homogenize all variables across all sites, we embraced the heterogeneity to better approximate clinical trials. Awareness of the heterogeneity, its sources, and how it impacts the study performance may further improve the study design and statistical modeling for future multicenter preclinical trials.

Diurnal Differences in Immune Response in Brain, Blood and Spleen After Focal Cerebral Ischemia in Mice.

The immune response to acute cerebral ischemia is a major factor in stroke pathobiology. Circadian biology modulates some aspects of immune response. The goal of this study is to compare key parameters of immune response during the active/awake phase versus inactive/sleep phase in a mouse model of transient focal cerebral ischemia. Mice were housed in normal or reversed light cycle rooms for 3 weeks, and then they were blindly subjected to transient focal cerebral ischemia. Flow cytometry was used to examine immune responses in blood, spleen, and brain at 3 days after ischemic onset. In blood, there were higher levels of circulating T cells in mice subjected to focal ischemia during zeitgeber time (ZT)1-3 (inactive or sleep phase) versus ZT13-15 mice (active or awake phase). In the spleen, organ weight and immune cell numbers were lower in ZT1-3 versus ZT13-15 mice. Consistent with these results, there was an increased infiltration of activated T cells into brain at ZT1-3 compared with ZT13-15. This proof-of-concept study indicates that there are significant diurnal effects on the immune response after focal cerebral ischemia in mice. Hence, therapeutic strategies focused on immune targets should be reassessed to account for the effects of diurnal rhythms and circadian biology in nocturnal rodent models of stroke.

Research advances in the application of vagus nerve electrical stimulation in ischemic stroke.

Stroke seriously endangers human well-being and brings a severe burden to family and society. Different post-stroke dysfunctions result in an impaired ability to perform activities of daily living. Standard rehabilitative therapies may not meet the requirements for functional improvement after a stroke; thus, alternative approaches need to be proposed. Currently, vagus nerve stimulation (VNS) is clinically applied for the treatment of epilepsy, depression, cluster headache and migraine, while its treatment of various dysfunctions after an ischemic stroke is still in the clinical research stage. Recent studies have confirmed that VNS has neuroprotective effects in animal models of transient and permanent focal cerebral ischemia, and that its combination with rehabilitative training significantly improves upper limb motor dysfunction and dysphagia. In addition, vagus-related anatomical structures and neurotransmitters are closely implicated in memory-cognition enhancement processes, suggesting that VNS is promising as a potential treatment for cognitive dysfunction after an ischemic stroke. In this review, we outline the current status of the application of VNS (invasive and non-invasive) in diverse functional impairments after an ischemic stroke, followed by an in-depth discussion of the underlying mechanisms of its mediated neuroprotective effects. Finally, we summarize the current clinical implementation challenges and adverse events of VNS and put forward some suggestions for its future research direction. Research on VNS for ischemic stroke has reached a critical stage. Determining how to achieve the clinical transformation of this technology safely and effectively is important, and more animal and clinical studies are needed to clarify its therapeutic mechanism.

Intranasal Delivery of RGD-Containing Osteopontin Heptamer Peptide Confers Neuroprotection in the Ischemic Brain and Augments Microglia M2 Polarization.

Osteopontin (OPN), a phosphorylated glycoprotein, is induced in response to tissue damage and inflammation in various organs, including the brain. In our previous studies, we reported the robust neuroprotective effects of the icosamer OPN peptide OPNpt20, containing arginine-glycine-aspartic acid (RGD) and serine-leucine-alanine-tyrosine (SLAY) motifs, in an animal model of transient focal ischemia and demonstrated that its anti-inflammatory, pro-angiogenic, and phagocytosis inducing functions are responsible for the neuroprotective effects. In the present study, we truncated OPNpt20 to 13 or 7 amino acid peptides containing RGD (R) and/or SLAY (S) motifs (OPNpt13RS, OPNpt7R, OPNpt7RS, and OPNpt7S), and their neuroprotective efficacy was examined in a rat middle cerebral artery occlusion (MCAO) model. Intranasal administration of all four peptides significantly reduced infarct volume; OPNpt7R (VPNGRGD), the 7-amino-acid peptide containing an RGD motif, was determined to be the most potent, with efficacy comparable to that of OPNpt20. Additionally, sensory–motor functional deficits of OPNpt7R-administered MCAO animals were significantly improved, as indicated by the modified neurological severity scores and rotarod test. Notably, the expression of M1 markers was suppressed, whereas that of M2 markers (Arginase 1, CD206, and VEGF) was significantly enhanced in OPNpt7R-treated primary microglia cultures. Inflammation resolution by OPNpt7R was further confirmed in MCAO animals, in which upregulation of anti-inflammatory cytokines (Arg1, IL-10, IL-4, and CD36) and enhanced efferocytosis were detected. Moreover, studies using three mutant peptides (OPNpt7R-RAA or OPNpt7R-RAD, where RGD was replaced with RAA or RAD, respectively, and OPNpt7R-sc containing scrambled sequences) revealed that the RGD motif plays a vital role in conferring neuroprotection. In conclusion, the RGD-containing OPN heptamer OPNpt7R exhibits neuroprotective effects in the post-ischemic brain by suppressing M1 markers and augmenting M2 polarization of microglia and the RGD motif plays a critical role in these activities.

Acute ischemic stroke triggers a cellular senescence-associated secretory phenotype

Senescent cells are capable of expressing a myriad of inflammatory cytokines and this pro-inflammatory phenomenon is known as senescence-associated secretory phenotype (SASP). The contribution of this phenomenon in brain ischemia was scarce. A mouse model of transient focal cerebral ischemia by compressing the distal middle cerebral artery (tMCAo) for 60 min was used. SASP, pro-inflammatory cytokines and cell cycle mRNAs levels were quantified at 30-min and 72 h post-surgery. Immunohistochemistry in paraffin embedded human brain slides and mouse brain tissue was performed. Our results showed an increase of both p16 and p21 mRNA restricted to the infarct area in the tMCAo brain. Moreover, there was an induction of Il6, Tnfa, Cxc11, and its receptor Cxcr2 mRNA pro-inflammatory cytokines with a high positive correlation with p16/p21 mRNA levels. The p16 was mainly shown in cytoplasm of neurons and cytoplasm/membrane of microglial cells. The p21 was observed in membrane of neurons and also it showed a mixed cytoplasmic and membranous pattern in the microglial cells. In a human stroke patient, an increase of P16 in the perimeter of the MCA infarct area was observed. These suggest a role of SASP in tMCAo mouse model and in human brain tissue. SASP potentially has a physiological role in acute ischemic stroke and neurological function loss.

Synthesis and biological evaluation of 1,2,4-oxadiazole core derivatives as potential neuroprotectants against acute ischemic stroke

Here, we report the synthesis and neuroprotective capacity of 27 compounds with a bisphenol hydroxyl-substituted 1,2,4-triazole core or 1,2,4-oxadiazole core for stroke therapy. In vitro studies of the neuroprotective effects of compounds 1–27 on sodium nitroprusside (SNP)-induced apoptosis in PC12 cells indicate that compound 24 is the most effective compound conferring potent protection against oxidative injury. Compound 24 inhibits reactive oxygen species (ROS） accumulation and restores the mitochondrial membrane potential (MMP). Moreover, further analysis of the mechanism showed that compound 24 activates the antioxidant defence system by promoting the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and increasing the expression of haem oxygenase 1 (HO-1). An in vivo study was performed in a rat model of transient focal cerebral ischaemia generated by the intraluminal occlusion of the middle cerebral artery (MCAO). Compound 24 significantly reduced brain infarction and improved neurological function. Overall, compound 24 potentially represents a promising compound for the treatment of stroke.

Systemic administration of sunflower oil exerts neuroprotection in a mouse model of transient focal cerebral ischaemia.

Natural products are valuable sources of nutraceuticals for the prevention or treatment of ischemic stroke, a major cause of death and severe disability worldwide. Among the mechanisms implicated in cerebral ischemia-reperfusion damage, oxidative stress exerts a pivotal role in disease progression. Given the high antioxidant potential of most components of sunflower oil, we have explored its effects on ischemic brain injury produced in the mouse by transient occlusion of the middle cerebral artery (MCAo). Intraperitoneal (i.p.) administration of sunflower oil at doses of 3 ml/kg (48 h, 24 h and 1 h before MCAo) significantly reduced brain infarct volume and oedema assessed 24 h after the insult. This neuroprotective treatment schedule also prevented the elevation of brain lipid peroxidation produced by MCAo-reperfusion injury. By contrast, doses of 0.03 ml/kg of sunflower oil resulted ineffective on both cerebral damage and lipid peroxidation. Although sunflower oil did not affect serum levels of Diacron-reactive oxygen metabolites (d-ROMs), both 0.03 and 3 ml/kg dosing regimens resulted in the preservation of serum biological antioxidant potential (BAP) that was otherwise dramatically reduced 24 h after MCAo. Sunflower oil represents a promising source of neuroprotective extracts/compounds that can be exploited for the prevention and/or treatment of cerebral ischemia.

SMI-32 — a novel axonal injury marker for investigation of ischemic brain pathology

The relevance of this work is determined by the high prevalence and social significance of cerebrovascular diseases and the need to develop effective methods for verifying neuronal damage due to cerebral ischemia in experimental models.
 The aim of this study was to assess the possibility of immunohistochemical revealing of neurofilaments to detect axonal injury in cerebral ischemia models.
 Materials and methods. A model of transient focal cerebral ischemia by the left middle cerebral artery occlusion was reproduced in male Wistar, SHR and WKY rats. Axonal injury was assessed by immunohistochemical reactions for neurofilament proteins using SMI-32 and 2F11 antibodies.
 Results. In cerebral ischemia, damage to nerve fibers occurs, manifested by thickening of axons, their varicose expansion and segmental accumulation of neurofilament proteins. These changes are more noticeable with an immunohistochemical reaction to the SMI-32 marker of neurofilament heavy chain.
 Conclusions. The use of antibodies to the non-phosphorylated neurofilament heavy chain makes it easy to identify degenerating nerve fibers and can be recommended as an alternative method for detecting axonal injury.

Cardiac functional consequences of stroke induced by transient middle cerebral artery occlusion in a rodent model

Abstract Background The cardiac functional consequences of ischaemic stroke are still need to be elucidated, while according to ethical issues only non-inasive measurents were carried out in patients underwent transient cerebral ischaemia. Purpose We aimed at investigating left ventricular function using non-invasive and invasive modalities in a rat model of transient focal ischaemia. Methods Age-matched, young adult rats were used for this study. Serial left ventricular echocardiographic measurements and speckle-tracking analysis were performed in rats (n=9) underwent transient middle cerebral artery occlusion (MCAO) before, during and immediately after the induction of stroke, with a follow-up at 24, 48, 72 hours; 7, 11 and 14 days. In another experimental setting, 48 hours after stroke induction (MCAO group, n=9) we characterized left ventricular function by pressure-volume analysis, that was compared to sham-operated controls (Co group, n=9). Results Serial echocardiographic measurements showed impaired systolic function, that was most severe 48 hours after MCAO (global circumferential strain, GCS: −14.8±2.6% 48 hours after MCAO vs. −19.3±2.4% baseline, p<0.05). A complete recovery of systolic functional deterioration was observed after 14 days (GCS: −19.2±2.5% 14 days after MCAO vs. −19.3±2.4% baseline, n.s.). Heart weight (normalized to tibial weight) did not differ between MCAO and Co animals. Pressure-volume analysis revealed unaltered diastolic function and showed unchanged load-independent contractility index values (slope of end-systolic pressure-volume relationship, ESPVR: 2.56±0.29mmHg/μl MCAO vs. 2.55±0.59 mmHg/μl Co, n.s.) after MCAO. There was a tendency towards increased systolic pressure and deteriorated ventriculo-arterial coupling in animals underwent stroke. Conclusions Our data suggests that MCAO is associated with reversible impairment of systolic function during echocardiographic measurements, however without alteration of intrinsic myocardial contractility. The tendency towards increased afterload might explain the observed alterations in rats underwent stroke. Funding Acknowledgement Type of funding source: None

Neuroprotective Effects of a Novel Inhibitor of c-Jun N-Terminal Kinase in the Rat Model of Transient Focal Cerebral Ischemia.

A novel specific inhibitor of c-Jun N-terminal kinase, 11H-indeno[1,2-b]quinoxalin-11-one oxime sodium salt (IQ-1S), has a high affinity to JNK3 compared to JNK1/JNK2. The aim of this work was to study the mechanisms of neuroprotective activity of IQ-1S in the models of reversible focal cerebral ischemia (FCI) in Wistar rats. The animals were administered with an intraperitoneal injection of IQ-1S (5 and 25 mg/kg) or citicoline (500 mg/kg). Administration of IQ-1S exerted a pronounced dose-dependent neuroprotective effect, not inferior to the effects of citicoline. Administration of IQ-1S at doses of 5 and 25 mg/kg reduced the infarct size by 20% and 50%, respectively, 48 h after FCI, whereas administration of citicoline reduced the infarct size by 34%. The administration of IQ-1S was associated with a faster amelioration of neurological status. Control rats showed a 2.0-fold increase in phospho-c-Jun levels in the hippocampus compared to the corresponding values in sham-operated rats 4 h after FCI. Administration of IQ-1S at a dose of 25 mg/kg reduced JNK-dependent phosphorylation of c-Jun by 20%. Our findings suggest that IQ-1S inhibits JNK enzymatic activity in the hippocampus and protects against stroke injury when administered in the therapeutic and prophylactic regimen in the rat model of FCI.

Brain Factor-7\xae improves learning and memory deficits and attenuates ischemic brain damage by reduction of ROS generation in stroke in vivo and in vitro

Brain Factor-7® (BF-7), silk fibroin peptide, is known to be effective in improvement of memory and learning ability. In this study, the effects of BF-7 (10 mg/kg, p.o., pre-treatment for 7 days and post-treatment for 7 days) on neuroprotection and memory and learning functions were investigated in a rat model of transient focal cerebral ischemia and a gerbil model of transient global forebrain ischemia. Furthermore, to find the mechanism of BF-7, we examined the neuroprotective and antioxidative effects of BF-7 in vitro using neuroblastoma (SH-SY5Y) cells. In vivo model, treatment with BF-7 significantly reduced the number of errors in 8-arm maze test and significantly increased latency time in passive avoidance test at 7 days after focal ischemia compared to those in the vehicle-treated group. In addition, treatment with BF-7 significantly decreased the infarct size or neuronal death at 7 day following transient ischemia compared to that in the vehicle-treated group. In vitro model, 10 or 20 μg/ml of BF-7 treatment significantly increased cell viability in dose-dependent manner. In addition, oxidative stress was significantly attenuated in the ischemic cells, showing that 10 or 20 μg/ml of BF-7 treatment significantly reduced the generation of reactive oxygen species (ROS) compared to that in the ischemic cells. These results indicate that BF-7 treatment can attenuate ischemic damages and improve memory deficits via reduction of ROS generation.

Ethyl Pyruvate Increases Post-Ischemic Levels of Mitochondrial Energy Metabolites: A 13C-Labeled Cerebral Microdialysis Study.

Mitochondrial dysfunction after transient cerebral ischemia can be monitored by cerebral microdialysis (CMD) using changes in the lactate and pyruvate concentrations and ratio. Other metabolites associated with mitochondrial (dys)function are, e.g., tricyclic acid (TCA) and purine metabolites. Ethyl pyruvate (EP) is a putative neuroprotectant, supposedly targeting mitochondrial energy metabolism, but its effect on cerebral energy metabolism has never been described using microdialysis. In this study we monitored the metabolic effects of EP in the endothelin-1 (ET-1) rat model using perfusion with 13C-succinate and analysis of endogenous and 13C-labeled metabolites in the dialysates by liquid chromatography-mass spectrometry (LC-MS). Adult Sprague Dawley rats (n = 27 of which n = 11 were included in the study) were subjected to the microdialysis experiments. Microdialysis probes were perfused with 13C-labeled succinate (1 mM), and striatal dialysates were collected at 30 min intervals before induction of the insult, during intracerebral application of ET-1, and during intravenous treatment with either EP (40 mg/kg) or placebo, which was administered immediately after the insult. The rats were subjected to transient cerebral ischemia by unilateral microinjection of ET-1 in the piriform cortex, causing vasoconstriction of the medial cerebral artery. Monitoring was continued for 5 h after reperfusion, and levels of endogenous and 13C-labeled energy metabolites before and after ischemia-reperfusion were compared in EP-treated and control groups. Infarct volumes were assessed after 24 h. In both the EP-treated and placebo groups, ET-1-induced vasoconstriction resulted in a transient depression of interstitial glucose and elevation of lactate in the ipsilateral striatum. In the reperfusion phase, the concentrations of labeled malate, isocitrate, and lactate as well as endogenous xanthine were significantly higher in the EP-group than in the placebo-group: (mean ± SEM) labeled malate: 39.5% ± 14.9, p = 0.008; labeled isocitrate: 134.8% ± 67.9, p = 0.047; labeled lactate: 61% ± 22.0, p = 0.007; and endogenous xanthine: 93.9% ± 28.3, p = 0.0009. In the placebo group, significantly elevated levels of uridine were observed (mean ± SEM) 32.5% ± 12.7, p = 0.01. Infarct volumes were not significantly different between EP-treated and placebo groups, p = 0.4679. CMD labeled with 13C-succinate enabled detection of ischemic induction and EP treatment effects in the ET-1 rat model of transient focal cerebral ischemia. EP administered as a single intravenous bolus in the reperfusion-phase after transient cerebral ischemia increased de novo synthesis of several key intermediate energy metabolites (13C-malate, 13C-isocitrate, and endogenous xanthine). In summary, mitochondria process 13C-succinate more effectively after EP treatment.

Hypertonic saline mediates the NLRP3/IL-1β signaling axis in microglia to alleviate ischemic blood-brain barrier permeability by downregulating astrocyte-derived VEGF in rats.

IntroductionThe aim of this study was to explore whether the antibrain edema of hypertonic saline (HS) is associated with alleviating ischemic blood‐brain barrier (BBB) permeability by downregulating astrocyte‐derived vascular endothelial growth factor (VEGF), which is mediated by microglia‐derived NOD‐like receptor protein 3 (NLRP3) inflammasome.MethodsThe infarct volume and BBB permeability were detected. The protein expression level of VEGF in astrocytes in a transient focal brain ischemia model of rats was evaluated after 10% HS treatment. Changes in the NLRP3 inflammasome, IL‐1β protein expression, and the interleukin‐1 receptor (IL1R1)/pNF‐кBp65/VEGF signaling pathway were determined in astrocytes.ResultsHS alleviated the BBB permeability, reduced the infarct volume, and downregulated the expression of VEGF in astrocytes. HS downregulates IL‐1β expression by inhibiting the activation of the NLRP3 inflammasome in microglia and then downregulates VEGF expression by inhibiting the phosphorylation of NF‐кBp65 mediated by IL‐1β in astrocytes.ConclusionsHS alleviated the BBB permeability, reduced the infarct volume, and downregulated the expression of VEGF in astrocytes. HS downregulated IL‐1β expression via inhibiting the activation of the NLRP3 inflammasome in microglia and then downregulated VEGF expression through inhibiting the phosphorylation of NF‐кBp65 mediated by IL‐1β in astrocytes.

Abstract TP100: Post-Transcriptional Inactivation of MMP-12 Immediately After Reperfusion Facilitates Neurological Recovery After Ischemic Stroke

Introduction: We recently showed in a rodent model of transient focal cerebral ischemia that matrix metalloproteinase-12 (MMP-12) induction in the ischemic brain promotes post-stroke blood-brain barrier disruption, apoptosis, demyelination, and infarction. The purpose of the present study is to investigate the role of elevated MMP-12 on post-stroke neurological function and to identify the time window of therapeutic opportunity for MMP-12 suppression. Methods: Adult male Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion and reperfusion. Cohorts of rats (n =8-15/group) were administered with either MMP-12 shRNA or scrambled shRNA sequence (vehicle control) expressing plasmids (1 mg/Kg; intravenous) formulated as nanoparticles. The differences in sample size of various cohorts were attributed to the exclusion criteria followed, high mortality rate in vehicle control-treated group, and sample size required for statistical analysis. To assess the reflex, balance, sensory, and motor functions, rats from various cohorts were subjected to modified neurological severity scoring (mNSS), adhesive removal test, beam walk test and rotarod test at day 1, 3 and 5 of reperfusion. To assess the time window of therapeutic opportunity, various cohorts of rats were treated at 5 min, 3h, and 6h of reperfusion. Investigators blinded to study groups analyzed all outcome parameters. Results: The post-stroke percent survival rate in cohorts treated with MMP12shRNA expressing plasmids range from 82 to 89 as compared to 67 in vehicle control-treated group. The cohort of rats treated at 5 min of reperfusion with MMP-12snRNA expressing plasmids showed significantly better functional recovery as assessed by various neurological tests. However, delayed administration of MMP-12snRNA expressing plasmid (either at 3h or 6h of reperfusion) failed to promote any significant improvement in post-stroke neurological recovery. Conclusions: Post-stroke induction of MMP-12 in the ischemic brain contributes to neurological deficits and impedes recovery. MMP-12 targeting treatments immediately after reperfusion could offer substantial therapeutic benefits.

Osteopontin heptamer peptide containing the RGD motif enhances the phagocytic function of microglia

Osteopontin (OPN) is a phosphorylated glycoprotein expressed in various tissues, including brain, and mediates a wide range of cellular activities. In our previous studies, we reported recombinant OPN and RGD and SLAY-containing OPN-peptide icosamer (OPNpt20) exhibited robust neuroprotective activities in an animal model of transient focal ischemia, and attributed these effects to the anti-inflammatory, pro-angiogenic, and phagocytic functions of OPNpt20. In the present study, we truncated OPNpt20 to 13 or 7 amino acid peptides containing RGD (R) and/or SLAY (S) motif (OPNpt13RS, OPNpt7R, OPNpt7RS, and OPNpt7S) and their cell motility and migration inducing activities were examined in BV2 cells (a microglia cell line). All four peptides significantly enhanced BV2 cell motility and migration, but OPNpt7R, an RGD-containing 7-amino-acid OPN peptide (VPNGRGD), was found to be most potent and its potency was comparable to OPNpt20. Phagocytic activity and F-actin polymerization were also significantly enhanced in OPNpt7R-treated BV2 cells. Importantly, studies using two mutant peptides (OPNpt7R-RAA and OPNpt7R-RAD, wherein RGD in OPNpt7R was replaced with RAA or RAD, respectively) revealed that all these effects of OPNpt7R, motility, migration, F-actin polymerization, and phagocytosis induction, were RGD-dependent. Furthermore, the Erk, Fak, and Akt signaling pathways appeared to be involved in the induction of phagocytic activity by OPNpt7R. Co-treating cells with OPNpt7R and D98059 or wortmannin (pharmacological inhibitors of Erk and Akt, respectively) significantly suppressed OPNpt7R-mediated phagocytosis induction. These results indicate the RGD-containing OPN heptamer OPNpt7R triggers microglial motility, migration, and phagocytic activity and that the RGD motif plays a critical role in these activities.